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(Received for publication, August 2, 1996)
From the Department of Chemistry, Biochemistry, and Molecular
Biology, Oregon Graduate Institute of Science & Technology,
Portland, Oregon 97291-1000
Translational control mediated by an upstream
open reading frame (uORF) in the 5
Volume 272, Number 1,
Issue of January 3, 1997
pp. 255-261
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
-leader of the Neurospora
crassa arg-2 mRNA was reconstituted in a homologous,
cell-free in vitro translation system. A cell-free N. crassa system was developed that required the presence of cap and
poly(A) on RNA for maximal translation and that was amino
acid-dependent. The 24-codon arg-2 uORF, when placed in the 5-leader region of capped and adenylated synthetic luciferase RNAs, conferred Arg-specific negative regulation in this
system. Improving the uORF translation initiation context decreased
luciferase production and only slightly increased the magnitude of
Arg-specific regulation. Mutation of uORF Asp codon 12 to Asn, which
eliminates Arg-specific regulation in vivo, eliminated regulation in vitro. Elimination of the uORF translation
initiation codon also eliminated Arg-specific regulation. Arg-specific
regulation in vitro appeared to be reversible. Control of
RNA stability did not appear to be a primary component of Arg-specific
regulation in vitro. Comparison of the effects of adding
Arg to in vitro translation reactions with adding compounds
related to Arg indicated that Arg-specific translational regulation was
specific for L-arginine.
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